Process for preparing macrolide antibiotics by culturing streptomyces hirsutus ATCC 53513

ABSTRACT

An antibiotic complex, containing three major components, has been isolated from fermentations of a new strain of the microorganism Streptomyces hirautus. The major components are new, neutral, macrolide antibiotic compounds, which are useful as antibacterial agents against certain gram-positive bacteria.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of application Ser. No. 06/938,219, filedDec. 5, 1986, now U.S. Pat. No. 4,835,141.

BACKGROUND OF THE INVENTION

This invention relates to a new antibiotic complex which has beenisolated by fermentation of a new strain of the microorganismStreptomyces hirsutus. The new antibiotic complex has been designatedCP-61,884, and it has been shown to contain three major components.Chemically, these major components are new members of the macrolideclass of antibiotics.

The three major components of antibiotic complex CP-61,884 are neutralmacrolides, containing a 16-membered lactone ring, and they can becompared to chalcomycin. See further, The Merck Index, 10th Edition,Merck & Co., Inc., Rahway, New Jersey, U.S.A., 1983, Abstract No. 1990(page 283).

SUMMARY OF THE INVENTION

This invention provides new macrolide antibiotic compounds selected fromthe group consisting of ##STR1## wherein R¹ is the radical of theformula ##STR2## and R² is a radical selected from the group consistingof ##STR3##

The macrolide compounds of the formulas I and II have been isolated fromfermentations of a new strains of the microorganism Streptomyceshirsutus, and said compounds are antibacterially-active against certaingram-positive bacteria. The new strain of Streptomyces hirsutus wasinitially designated N521-25, and it is now on deposit with the AmericanType Culture Collection under Accession No. 53513.

Accordingly, also included within this invention are: a method oftreating a bacterial infection in a mammalian subject using a compoundof formula I or II; pharmaceutical compositions which comprise acompound of the formula I or II and a pharmaceutically-acceptablecarrier; a process for preparing a compound of the formula I or II byfermentation of Streptomyces hirsutus ATCC 53513, or a mutant thereofhaving the ability to produce a compound of formula I or II; and abiologically-pure culture of Streptomyces hirsutus ATCC 53513, or amutant thereof having the ability to produce a compound of formula I orII.

The preferred macrolide antibiotic of this invention is the compound offormula I, wherein R¹ is of formula III and R² is of formula V.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of formulas I and II are the three major components of anew antibiotic complex which has been obtained by fermentations of a newmicroorganism of the genus Streptomyces. The new microorganism wasisolated from a soil sample collected from a woodland in Sacramento,California, and it was designated initially as culture N521-25.Subsequently, it was characterized and identified by Liang H. Huang,Ph.D., Pfizer Inc., Groton, Connecticut, as described herein below.Culture N521-25 was shown thereby to be a new strain of Streptomyceshirsutus.

Culture N521-25 was found to produce narrow hyphae of theActinomycetales, spore chains produced on the aerial mycelium and anunfragmented substrate mycelium, characteristic of the genusStreptomyces.

Culture N521-25 was planted from a slant into liquid, ATCC medium No.172 and grown for four days at 28° C. on a shaker. It was thencentrifuged for 20 minutes, washed three times with sterile distilledwater and planted onto media commonly used for identification of membersof the Actinomycetales. The cultures were incubated at 28° C. andresults were read at appropriate times, most-commonly at 14 days.Specific identification media used for the characterization of cultureN521-25, and references for their composition, are as follows:

1. Tryptone Yeast Extract Broth--(ISP #1 medium, Difco).

2. Yeast Extract-Malt Extract Agar--(ISP #2 medium, Difco).

3. Oatmeal Agar--(ISP #3 medium, Difco).

4. Inorganic Salts-Starch Agar--(ISP #4 medium, Difco).

5. Glycerol-Asparagine Agar--(ISP #5 medium, Difco).

6. Czapek-Sucrose Agar--S. A. Waksman, The Actinomycetes, Vol. 2, mediumno. 1, p. 328, 1961.

7. Glucose-Asparagine Agar--Ibid, medium no. 2, p. 328.

8. Bennett's Agar--Ibid, medium no. 30, p. 331.

9. Emerson's Agar--Ibid, medium no. 28, p. 331.

10. Nutrient Agar--Ibid, medium no. 14, p. 330.

11 . Peptone-Yeast Extract Iron Agar - (ISP #6 medium, Difco).

12. Gordon and Smith's Tyrosine Agar--R. E. Gordon and M. M. Smith, J.Bacteriol. 69: 147-150, 1955.

13. Casein Agar--Ibid.

14. Calcium Malate Agar--S. A. Waksman, Bacteriol. Rev. 21: 1-29, 1957.

15. Gelatin--R. E. Gordon and J. M. Mihm, J. Bacteriol. 73: 15-27, 1957.

16. Starch--Ibid.

17. Organic Nitrate Broth--Ibid.

18. Dextros Nitrate Broth--S. A. Waksman, The Actinomycetes, Vol. 2,medium no. 1, p. 328, 1961, with 3 g dextrose substituted for 30 gsucrose and agar omitted.

19. Potato Carrot Agar--M. P. Lechevalier, J. Lab. and Clin. Med. 71:934-944, 1968, but use only 30 g potatoes, 2.5 g carrot and 20 g agar.

20. 2% Tap Water Agar.

21. Skim Milk--Difco.

22. Cellulose utilization --(a)

H. L. Jensen, Proc. Linn. Soc. N.S.W. 55: 231-248, 1930.

(b) M. Levine and H. W. Schoenlein, A Compilation of Culture Media,medium no. 2511, 1930.

23. Carbohydrates--ISP #9 medium, Difco.

24. Temperature Range--ATCC medium 196 in ATCC Media Handbook lst ed.,p. 11, 1984.

Culture N521-25 exhibited the following characteristics, with colorsbeing given in common terminology and also with reference to color chipsfrom the Color Harmony Manual, 4th Edition. The methods of whole cellamino acid and sugar analyses were those described in Becker, B. et al.,Appl. Microbiol. 12: 421-423, 1964; and in Lechevalier, M. P., J. Lab.Clin. Med., 71: 934-944, 1968.

Yeast Extract-Malt Extract Agar--Growth good, white, cream to blue (2ca,near gray series 22dc, 24fe); raised, granular to wrinkled, with whiteto white-blue aerial mycelium; reversed yellowish to yellowish-brown(21c, 31c, 31e); no soluble pigment.

Oatmeal Agar--Growth poor to moderate, pale gray to pale green-gray tocream (2ca, near gray series ldc, 241/2dc, 24dc, 24fe); slightly raisedto raised, smooth to granular, or appearing as isolated colonies; aerialmycelium pale gray to pale green-gray; reverse pale gray to cream (neargray series 2bc, 2ca); no soluble pigment.

Inorganic Salts-Starch Agar--Growth moderate to good, cream, yellowish,yellowish brown to green-gray (2ca, 3ea, 31c, near gray series 24fe,241/2fe); slightly raised, smooth, or granular in some areas; aerialmycelium green-gray; reverse cream, yellowish brown to green-gray (2ca,3ic, near gray series 24fe); no soluble pigment.

Glycerol-Asparagine Agar--Growth good, white, pale yellow-orange, grayto green-gray (3ea, near gray series lfe, 241/2fe); raised, granular, orslightly wrinkled; aerial mycelium same as surface; reverse pale gray,yellowish to brown (near gray series ldc, 21c, 31g); soluble pigmentcream (2ca).

Czapek-Sucrose Agar--Growth moderate, pale green-gray, pale gray to gray(near gray series 241/2dc, 2dc, 2fe, 2ih); thin to slightly raised,smooth to granular; aerial mycelium same as surface; reverse pale grayto gray (near gray series 2dc, 2fe); no soluble pigment.

Glucose-Asparagine Agar--Growth poor to moderate, cream (2ca), slightlyraised; appearing as isolated, small colonies; aerial mycelium lacking;reverse cream (2ca); no soluble pigment.

Gordon and Smith's Tyrosine Agar--Growth moderate to good, green-graywith black sectors (near gray series 241/2ih, 24ih, 24ml), slightlyraised, smooth; aerial mycelium same as surface; reverse gray togreen-gray (near gray series lfe, 241/2fe); soluble pigment paleyellowish (2ea).

Calcium Malate Agar--Growth moderate, white, pale pink to dark gray(4ca, near gray series lih, lml); thin or raised, smooth or granular;aerial mycelium same as surface; reverse pink to gray (5ca, near grayseries 2fe); no soluble pigment.

Casein Agar--Growth good, brown to dark brown (4ie, 41g), moderatelyraised, wrinkled or granular, with sectors of white aerial mycelium;reverse same as surface; soluble pigment brown (41c).

Bennett's Agar--Growth good, white, cream to orange (2ca, 4ia, 41c);moderately raised, slightly wrinkled, with white aerial mycelium;reverse cream to yellowish (2ca, 3ga); no soluble pigment.

Emerson's Agar--Growth good, white to green-gray (near gray series241/2fe, 241/2ih, 24ih), raised, wrinkled; aerial mycelium same assurface; reverse yellOwish brown to brown (41c, 31e); soluble pigmentyellowish brown (31c).

Nutrient Agar--Growth moderate to good, white to cream (2ca), moderatelyraised, smooth or slightly granular, with white aerial mycelium; reversecream to pale yellowish (2ca, 2ea); no soluble pigment.

Gelatin Agar--Growth good, pale green-gray (near gray series 241/2dc,24dc, 24fe) with a cream to red-purple edge (2ca, 61/2ie), moderatelyraised, wrinkled or granular but smooth toward the edge, with palegreen-gray aerial mycelium; reverse cream to brown (2ca, 41e, 41g); nosoluble pigment.

Starch Agar--Growth good, white, green-gray to brown (near gray series24fe, 24ih, 4ng) with a cream (2ca) edge; raised, wrinkled, with whiteto green-gray aerial mycelium; reverse brown (4ng, 4ne); no solublepigment.

Potato Carrot Agar--Growth moderate, green-gray (near gray series241/2fe, 241/2ih), slightly raised, smooth or granular, with green-grayaerial mycelium; reverse same as surface; no soluble pigment.

Tap Water Agar--Growth poor to moderate, green-gray (near gray series241/2fe, 241/2ih), thin to slightly raised, smooth, with green-grayaerial mycelium; reverse same as surface; no soluble pigment.

Morphological Properties--The morphological properties were observed oninorganic salts-starch agar after 14 days of incubation; spore mass inthe Green-color series; spore chains in Section Rectiflexibiles,straight, flexuous, wavy to hooked; short, 4 to 12 spores per sporechain; sporophores monopodially or verticillately branched; sporesglobose, oval to elliptical, 1.2-1.8 micrometer in diameter or 1.2-1.8(-2.4)×1.2-1.4 (-1.8) micrometer; spiny, as revealed by scanningelectron microscopy.

Biochemical Properties--Melanin not produced; hydrogen sulfide produced;gelatin liquefied; starch hydrolyzed; nitrate not reduced to nitrite;growth but no decomposition on both cellulose broths; coagulation andclearing on milk; casein digestion positive; calcium malate digestionpositive; tyrosine digestion positive. Carbohydrate utilization;glucose, arabinose, fructose, inositiol, mannitol, raffinose, rhamnose,sucrose, and xylose all utilized.

Cell-Wall Analyses--The whole-cell hydrolyzates were found to containLL-diaminopimelic acid and glucose.

The relationship of temperature to growth rate for culture N521-25 wasas follows: 21° C., moderate to good growth; 28° C., good growth; 37°C., moderate growth; and 45° C., poor growth.

The culture N521-25 is characterized by the negative melanin reaction,the green-gray aerial mycelium, the straight to flexuous spore chains,and the spores with a spiny surface. The whole-cell hydrolysates revealthe presence of LL-diaminopimelic acid and the absence of diagnosticsugars. The culture utilizes glucose, arabinose, fructose, inositol,mannitol, raffinose, rhamnose, sucrose and xylose. Compared with theknown species of Streptomyces, it closely resembles S. hirsutus. Thus,S. hirsutus ATCC 19773 was grown side-by-side and compared with cultureN521-25.

Except for its inability to reduce nitrate to nitrite, the cultureN521-25 agrees with S. hirsutus in all of the other biochemicalproperties as well as in the temperature relations. Both cultures sharethe same morphological properties and most of the cultural properties. Afew cultural differences were noted, however. The culture N521-25 butnot S. hirsutus may produce yellowish brown substrate mycelium on yeastextract-malt extract agar, inorganic salts-starch agar andglycerol-asparagine agar. In the culture N521-25, more aerial myceliumis produced on glycerol-asparagine agar, Czapek-sucrose agar andtyrosine agar. On some media such as inorganic salts-starch agar,Emerson's agar, potato carrot agar, and tap water agar, the aerialmycelium of the culture N521-25 shows slightly more greenish tint in thegreen-gray background than that of S. hirsutus. On Bennett's agar andcasein agar, the colonies of the culture N521-25 are cream to orange andbrown to dark brown, respectively, but those of S. hirsutus are cream.These cultural differences are considered as minor and it is concludedthat the culture N521-25 represents a new strain of Streptomyceshirsutus Ettlinger, Corbaz and Hutter.

Culture N521-25 was deposited with the American Type Culture Collection("ATCC"), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. under theprovisions of the Budapest Treaty on July 1, 1986, under Accession No.ATCC 53513. All restrictions on the availability of Streptomyceshirsutus ATCC 53513 will be irrevocably removed not later than the dateof issuance of a patent on this application. Additionally, thepermanancy of the deposit is guaranteed throughout the life of thepatent or for 30 years from the date of deposit, whichever is longer.

The antibiotic complex of this invention can be obtained by fermentingthe microorganism Streptomyces hirsutus ATCC 53513 under submergedaerobic conditions, followed by extraction of the complex from thefermentation broth. The compounds of formulas I and II can be obtainedfrom the complex by classical methods such as chromatography orcounter-current distribution. Moreover, a compound of formula I or IIcan be obtained by fermentation of a mutant of S. hirsutus ATCC 53513having the ability to produce a compound of formula I or II. Mutants ofS. hirsutus ATCC 53513 can be obtained by standard techniques and theycan arise naturally as a result of spontaneous mutations.

Fermentation of S. hirsutus ATCC 53513 or a mutant thereof can becarried out in a manner similar to that described in U.S. Pat. No.4,411,892 for fermentation of Streptomyces albus subsp. indicus ATCC39012. In general, the S. hirsutus can be grown from 24° to 36° C. undersubmerged conditions in an aqueous nutrient medium, with aeration andagitation. Suitable nutrient media contain an assimilable carbohydratesource, such as a sugar, starch or glycerol; organic nitrogensubstances, such as soybean mean, casamino acids or yeast extract;growth substances, such as grain solubles, fish meal or cotton seedmeal; mineral salts containing trace elements, such as iron, cobalt,copper and zinc; and calcium carbonate and/or phosphates as bufferingagents.

Inoculum is prepared by scraping vegetative cells from slants or Rouxbottles inoculated with Streptomyces hirsutus ATCC 53513 or a mutantthereof. A solid medium suitable for initial growth on slants and Rouxbottles is ATCC medium No. 172, which has the following composition.

    ______________________________________                                        ATCC 172                                                                                          Amount                                                    Ingredient          (gms./liter)                                              ______________________________________                                        Glucose             10                                                        Soluble Starch      20                                                        Yeast Extract        5                                                        NZ Amine A (Humko)*  5                                                        Calcium Carbonate    1                                                        Distilled Water to 1000 ml.;                                                  ph to 7.0 with KOH                                                            Add Agar            20                                                        ______________________________________                                         *A purified enzymatic digest of casein.                                  

Vegetative cells from slants are used to inoculate either shake flasksor inoculum tanks. Alternately the inoculum tanks are inoculated fromshake flasks. In shake flasks, growth will generally have reached itsmaximum in 96 to 120 hours, whereas in the inoculum tanks growth willusually be at the most favorable period in 72 to 96 hours afterinoculation. A fermentor is inoculated with vegetative broth from theinoculum flask or tank under completely aseptic conditions, andfermented for a period of 48 to 120 hours. Aeration is maintained in theshake flask by agitation on a shaker or in tanks by forcing sterile airthrough a sparger at the rate of 1/2to 2 volumes of air per volume ofbroth per minute. The speed of agitation (stirring) depends upon thetype of agitator employed; a shake flask is usually run at 150 to 200cycles per minute and a fermentor at 300 to 1700 revolutions per minute.Sterility is maintained at all times. The temperature is regulatedbetween 28° and 36° C. Foaming can be controlled using sterileantifoaming agents, such as refined soybean oil.

Shake flasks are prepared using one of the following media:

    ______________________________________                                        CL13NZ            JDYTT                                                                   Grams/                  Grams/                                    Ingredient  liter     Ingredient    liter                                     ______________________________________                                        Glucose     20        Cerelose      10                                        Soy Flour   10        Corn Starch   5                                         NZ Amine YTT*                                                                             5         Corn Steep Liquor                                                                           5                                         Sodium Sulfate                                                                            0.5       NZ Amine YTT* 5                                         Cobalt Chloride                                                                           0.002     Cobalt Chloride                                                                             0.002                                     Calcium Carbonate                                                                         2         Calcium Carbonate                                                                           3                                         Water to 1 liter;     Water to 1 liter;                                       ph 6.9-7.0            ph 6.9-7.0                                              ______________________________________                                         *An enzymatic digest of casein.                                          

One hundred milliliters of medium is distributed into 300 ml shakeflasks and sterilized at 120° C. and 15 p.s.i. for 30 minutes. Aftercooling, the medium is inoculated with a vegetative cell suspension fromthe Streptomyces hirsutus grown on ATCC 172 medium in agar. The flasksare shaken at 28° C. on a rotary shaker having a displacement of 1.5 to2.5 inches and 150 to 200 cycles per minute (CPM) for three to fourdays. One flask is used to inoculate a five liter fermentation vesselcontaining three liters of CL13NZ, JDYTT or the following medium:

    ______________________________________                                        CN-2                                                                          Ingredient       Grams/liter                                                  ______________________________________                                        Cerelose         10                                                           Corn Starch      10                                                           Soybean Flour    10                                                           NZ Amine YTT*    10                                                           Cobalt Chloride  0.002                                                        Calcium Carbonate                                                                              1                                                            Water to 1 liter;                                                             ph 6.9-7.0                                                                    ______________________________________                                         *An enzymatic digest of casein.                                          

One milliliter of L61 silicone is added as an antifoaming agent, thenthe vessels are sealed and sterilized at 120° C. and 15 p.s.i. for 45minutes. The pots are inoculated with one (ca 3% inoculum) flask,fermented for 96 to 144 hours at 30° C., stirred at 1700 revolutions perminute (RPM) with an air rate of one volume of air per volume of liquidper minute. When the fermentation is complete (based on an antibioticdisc assay using Staphylococus aureus ATCC 6538), the fermentors arestopped and extracted twice with one-third to one-half volume of asolvent, such as methyl isobutyl ketone or n-butanol. The solvent layeris separated from the aqueous phase by aspiration or centrifugation,sparkled, and concentrated in vacuo to a viscous oil.

The progress of antibiotic production during fermentation, and thebioactivity of the fermentation broth and recovery streams, can bemonitored by biological assay of the broth employing a sensitive strainof Micrococcus luteus (e.g., ATCC 9341) or Staphylococcus aureus (e.g.,ATCC 6538). The components in the broth and recovery streams can bevisualized by thin-layer chromatography (tlc) using Analtech silica 9elGF plates in ethyl acetate or chloroform/methanol(9:1). The developedplates are sprayed with vanillin reagent (3 g vanillin in 75 ml ethanoland 25 ml 85% phosphoric acid) and heated to 80° C. The antibiotics ofthis invention appear as greyish/blue spots. The developed tlc plate canalso be overlayed with agar, seeded with either S. aureus or M. luteusto which tetrazolium dye has been added, and incubated at 37° C. for 16hours to visualize the antibiotics (white against a pink background).

The antibiotic complex of this invention can be isolated afterfermentation of Streptomyces hirsutus ATCC 53513, or a mutant thereof,by extraction of the whole broth at natural pH with a volatile, organicsolvent, such as n-butanol, methyl isobutyl ketone or chloroform.Evaporation of the organic solvent then affords a syrup, from which theantibiotic complex, and ultimately the individual components thereof,can be recovered by repeated chromatography, especially using silicagel. Alternatively the whole broth can be filtered to remove themycelium, and then the filtrate is extracted, evaporated andchromatographed.

The antibiotic complex of this invention, and the individual componentsthereof of formulas I and II, possess antibacterial activity againstcertain gram-positive bacteria. This antibacterial activity can bedemonstrated by measuring the minimum inhibitory concentration (MIC) ofthe complex, or individual component thereof, against a variety oforganisms, according to standard procedures. Thus, the MIC's can bemeasured by the procedure recommended by the International CollaborativeStudy on Antibiotic Sensitivity Testing (Ericcson and Sherris, ActaPathologica et Microbiologia Scandinav, Supp. 217, Section B: 64-68[1971]), which employs brain heart infusion (BHI) agar and the inoculareplicating device. Overnight growth tubes are diluted 100 fold for useas the standard inoculum (20,000-10,000 cells in approximately 0.002 ml.are placed on the agar surface; 20 ml. of BHI agar/dish). Twelve 2 folddilutions of the test compound are employed, with initial concentrationof the test drug being 50 mcg./ml. Single colonies are disregarded whenreading plates after 18 hours at 37° C. The susceptibility (MIC) of thetest organism is accepted as the lowest concentration of compoundcapable of producing complete inhibition of growth as judged by theunaided eye. In particular the compounds of formulas I and II areantibacterially-active against certain strains of Staphylococcus aureusand Staphylococcus epidermidis.

The antibacterial activity of the antibiotic complex of the invention,and the individual components of formulas I and II, makes them valuablefor the treatment of bacterial infections caused by susceptibleorganisms in mammalian subjects, e.g., humans.

When using an antibacterial compound of this invention in a mammal, thecompound can be administered either alone, or in admixture with otherantibiotic substances and/or pharmaceutically-acceptable carriers ordiluents. Said carrier or diluent is chosen on the basis of the intendedmode of administration. For example, when considering an oral mode ofadministration, an antibacterial compound of this invention can be usedin the form of syrups, elixirs, aqueous solutions and suspensions, andthe like, in accordance with standard pharmaceutical practice. Theproportional ratio of active ingredient to carrier will naturally dependon the dosage contemplated; however, said proportional ratio willnormally be in the range from 2:1 to 1:5 by weight, and preferably 1:1to 1:4. An antibacterial compound of this invention can also beadministered parenterally, which includes intramuscular,intraperitoneal, subcutaneous and intravenous administration. For thesepurposes, sterile solutions of the active ingredient are usuallyprepared, and the pH of the solutions are suitably adjusted andbuffered. For intravenous use, the total concentration of solutes shouldbe controlled to render the preparation isotonic.

When a compound of the formula I or II is used to treat a bacterialinfection in a mammalian subject, an antibacterially-effective amountwill be used. Such an amount will not differ significantly from thedosages usually used for other macrolide antibiotics. In general, theprescribing physician or veterinarian will determine the appropriatedosage for a given host, and this will vary according to weight andresponse of the individual host, as well as the nature and severity ofthe host's symptoms and the virulence of the invading microorganism.However, in human subjects, the compounds of formula I and II willnormally be used orally at dosages in the range from about 20 to about50 milligrams per kilogram of body weight per day, and parenterally atdosages from about 10 to about 30 milligrams per kilogram of body weightper day, usually in divided dosages.

The following example is provided solely for further illustration.

EXAMPLE I Fermentation of Streptomyces hirsutus ATCC 53513.

Scale-up in large fermentors was carried out by preparing shake flaskscontaining 0.7 liters of CL13NZ or JDYTT medium. The shake flaskinoculum was fermented for 3 to 5 days at 28° C., and used to inoculatea 50 gallon fermentor containing 25 gallons of JDYTT medium.Approximately one liter of inoculum was used in the tank. The fermentor,after running 5 to 7 days, was harvested (ca. 25 gallons). The wholebroth was extracted with 1/5 volume of methyl isobutyl ketone at naturalpH, separated on an alpha DeLaval LAPX separator and the solvent wasconcentrated in vacuo to an oil. The oil was further concentrated on acyclone evaporator to a syrup. The syrup was suspended in heptane toremove the oils, and then it was filtered through a bed of filter aideand washed repeatedly with heptane. The antibiotic complex was dissolvedin chloroform, and then it was chromatographed on a silica gel column.The column was eluted sequentially with chloroform, chloroform/ethylacetate, ethyl acetate, acetone and lastly methanol. The elution wasfollowed by thin layer chromatography and bioassay of the fractions. Theactive cuts were combined, concentrated, dissolved in chloroform andrechromatographed on 150 g of Woelm alumina to give a purified solid.Additional separations were performed on LH20 sephadex in methanol,followed by chromatography by HPLC on a Waters Prep 500A silica gelcolumn using ethyl acetate. Finally, preparative thin-layerchromatography yielded the individual antibiotics of formulas I and II.

The ¹³ C nuclear magnetic resonance spectrum (CDC1₃ solution, 62.5 MHz)of the compound of the formula I, wherein R¹ is formula III and R² isformula V, showed absorptions at 200.95, 165.39, 151.21, 143.96, 125.60,120.55, 103.44, 100.93, 86.96, 81.92, 80.52, 79.69, 75.11, 72.74, 70.71,68.70, 67.82, 67.04, 61.69, 59.65, 59.04, 58.91, 56.81, 49.48, 44.68,41.78, 36.85, 34.14, 32.02, 20.91, 18.75, 18.41, 17.78, 17.58 and 17.01ppm, downfield from tetramethylsilane. On combustion analysis, thiscompound afforded the following results: C,61.27; H,8.19% (Calcd. forC₃₅ H₅₆ O₁₃ : C,61.40; H,8.23%).

We claim:
 1. A process for preparing a macrolide antibiotic compoundselected from the group consisting of ##STR4## wherein R¹ is the radicalof the formula ##STR5## and R² is a radical selected from the groupconsisting of ##STR6## which comprises cultivating the microorganismStreptomyces hirsutus ATCC 53513 or a mutant thereof having the abilityto produce a macrolide antibiotic compound selected from said group, inan aqueous culture medium containing assimilable sources of carbon,nitrogen and inorganic salts, under submerged, aerobic conditions, untila recoverable amount of a macrolide antibiotic compound selected fromsaid group is obtained.
 2. The process according to claim 1, whichcomprises cultivating the microorganism Streptomyces hirsutus ATCC 53513in an aqueous culture medium containing assimilable sources of carbon,nitrogen and inorganic salts, under submerged, aerobic conditions, untila recoverable amount of a macrolide antibiotic compound according toclaim 1 is obtained.
 3. A biologically-pure culture of the microorganismStreptomyces hirsutus ATCC 53513 or a mutant thereof, said microorganismor mutant thereof having the ability to produce a macrolide antibioticcompound according to claim 1, in recoverable quantity, upon cultivationin an aqueous culture medium containing assimilable sources of carbon,nitrogen and inorganic salts.
 4. A biologically-pure culture of themicroorganism Streptomyces hirsutus ATCC 53513, according to claim 3.